ROUNDTABLE DISCUSSION
Challenges and Opportunities in Epigenetic Drug Discovery Moderator: Richard T. Cummings1 Participants: Karen Maegley,2 W. Adam Hill,3 Robert Sims,4 and Sophie Mohin5 1
Senior Director, Lead Discovery, and 4Senior Director of Biology, Constellation Pharmaceuticals, Cambridge, Massachusetts. 2 Associate Research Fellow, Oncology Research Unit, Pfizer, San Diego, California. 3 Director, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts. 5 Assistant Managing Editor, Mary Ann Liebert, Inc., New Rochelle, New York.
Richard T. Cummings: Welcome to this roundtable discussion on epigenetic drug discovery. We have three participants: Karen Maegley from Pfizer in San Diego; Adam Hill from Novartis in Cambridge, Massachusetts; and Rob Sims from Constellation Pharmaceuticals in Cambridge, Massachusetts. Let us start with a general question, and then drill down a bit. We will begin with the question: What is your view on the current state of epigenetic drug discovery? Rob, please kick it off. Robert Sims: I will take a historical look back, at least from our perspective at Constellation Pharmaceuticals. We have been around for a little more than six years. When we were discussing the outlook for epigenetic drug discovery with our venture-backed support, several people questioned whether it was the right time or if it was too early. There were a number of companies that had already invested in epigenetics, for example, Novartis, because of the potential application in a variety of therapeutic areas. If you fast-forward six years later, I think the community as a whole, both in industry and in academia, has made tremendous progress. Perhaps the most important recent progress has been the observation of multiple epigenetic modalities that are beginning to show hints of clinical activity. From that perspective alone, there has probably never been a more exciting time to be in the epigenetic space. That is interesting from a number of different paradigms: writers, readers, as well as targets like IDH1 and IDH2, which very clearly impact the epigenetic state of cancers that have mutations in those targets and very robustly affect the methylation state of cells. Again, this reveals very interesting early signs of clinical activity. It is very exciting, and I think the next question at a high level is: Will this be sustainable over the next 5–10 years?
DOI: 10.1089/adt.2014.1502
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Richard T. Cummings: Karen? Karen Maegley: I am relatively new to this space compared to my colleagues at Constellation. I have been working in the epigenetic space for only a couple of years. But I completely agree with what was just said. In the early days (even from my perspective, which was late days from the Constellation perspective), there were many questions about whether we were getting in too early and whether we knew enough about the biology to really know which targets to choose. The industry as a whole (Epizyme, Constellation, GlaxoSmithKline, and others) has helped us in terms of the general acceptance of epigenetics as a therapy—for us, specifically in oncology, which is where I work. It is a really exciting time because it is still very new, and we do not know a lot about how to actually use these molecules. But we have definitely come a long way. Richard T. Cummings: And lastly, Adam? W. Adam Hill: Epigenetics is an emerging field. If you thought about regulating proteins and their activity 20-plus years ago, you would have been talking about kinases. There were some good breakthroughs in that area, which led to a large number of companies focusing on kinases as targets. I see epigenetic targets as one of the next generation of target classes, to be able to regulate proteins within the cell. From that perspective, I think there are some proven examples. There is a lot of chemical matter going through the clinic. Behind that, there is a lot of fundamental research going on. I see epigenetics as a burgeoning target class, which over the next few years will hopefully lead to drugs that make a real impact in patients’ lives. Richard T. Cummings: Following up on Adam’s comment about this being an emerging class, are there challenges associated with epigenetic targets that are unique to them relative to other, more traditional classes, such as G-protein coupled receptors (GPCRs) or kinases? If there are challenges, how have your teams addressed them? Rob, please start us off on this. Robert Sims: There are many challenges associated with the space, although, in general, most areas have challenges that are unique to them. You are planning to touch on this a little bit later, so I will skip the component about enzyme kinetics and the delayed response in cells. That is highly relevant to the epigenetic space when trying to monitor target engagement as well as phenotypic responses. Understanding how those molecular changes are manifested over time has been challenging, and it is something that the field has made progress on. I will use that as one example of the challenges and complexity of the epigenetic space.
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My scientific background has been rooted in chromatin and transcription. As we entered this particular field from a drug discovery perspective, like most things, we underestimated the complexity that would be visualized once we began to selectively and potently inhibit some of these chromatin coregulators. Epigenetic targets are not like kinases, for example, where you have an inhibitor and it inhibits catalytic activity (e.g., phosphorylation) of a particular kinase target. That is something you can rely on, although obviously there are complexities around that. What we found with bromodomain and extra terminal (BET) is that if you look in different cell contexts, the localization or the pattern of BET binding throughout the genome is quite different from cell to cell. Moreover, when you change or stimulate that cell, even in a one-cell context, the localization and distribution of that protein changes, and it can change over time. We have done experiments looking at very early transcriptional changes, minutes after treatment with BET inhibitors. That response is very dynamic over time, and it redistributes over time. In a way, it is a moving target. Trying to understand how inhibition of chromatin binding impacts a transcriptional program, even in one context over many dimensions, is complicated. But then trying to translate that into something practical, like how do I define predictive biomarkers of response in a patient population, just underscores the complexity and the challenge of dealing with these types of targets in this particular field. We and others in the field have made progress in understanding that, but it is absolutely a challenge when thinking about modulators of oncogenes as opposed to targeting oncogenes themselves. It is great that we know about the activating mutations of EZH2, but it has activity outside of that. Understanding that, how do you identify the populations that will best benefit from that mechanism? That is a challenge that obviously will have to be addressed over the next few years in trying to make these particular therapies useful to patients. Richard T. Cummings: Adam? W. Adam Hill: Rob touched on a number of good points. One difficulty we found when we first entered this field was working out exactly what a relevant assay was. You could go for a very simplistic form, for example, just looking at a catalytic activity. But when trying to put that into context and understand the different modes by which you could actually interrupt the activity, the numbers started to get much larger, and the ability to prove which assay was giving you the relevant information was quite tricky. So challenges include picking the target and working out how to make a biochemical assay for it, or looking at a cell-based assay and determining what an appropriate readout for that would be. Even if you are going into a phenotype, it is a challenge trying to define what that phenotype should be, thinking about three-dimensional cell culture versus two-dimensional, things like that. I think they all play into getting some more fundamental understanding of the biology around these targets, which allows you to put the appropriate assays together. With GPCRs or kinases, we have been in the field long enough to have a good handle on some of the quirks and nuances around those
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target families. Going into a new space like epigenetics, we have to come up, again, with a new toolkit, a new set of ways to go after these targets. Richard T. Cummings: Karen, have you found your experience in line with the other participants’? Karen Maegley: Absolutely. To expand on a couple of those things, with respect to a pharmacodynamic biomarker, for example, in a kinase, it is pretty straightforward to set up a cell-based assay to look at something like autophosphorylation or phosphorylation of the next kinase down the signaling pathway. But for some of these epigenetic targets, for example, a methyltransferase, monitoring the changes in the methyl mark at a global level will not always give you the information you need for your specific target. Sometimes it does and sometimes it doesn’t. Just figuring out what your cell-based readout is going to be can be quite challenging, depending on the target. Going back to the comment about a biologically relevant assay, if you want to use a nucleosome as a substrate, for example, it can be difficult to prepare with the quality that is necessary to do good biochemistry. If you are not using recombinant mononucleosomes, the oligonucleosome structure is very dynamic and very changeable, depending on what additives are in your assay buffer. Those are things that you really need to pay attention to. Using a peptide substrate is frequently not a very good surrogate, in my opinion, for a methyltransferase substrate. But from the biochemistry side of things, druggability seems to be a huge problem for us. With the BET inhibitors, that has not been an issue, but it seems to me, particularly in the methyltransferase space, that the number of solutions, the number of molecules that we have in our various collections that will be real inhibitors of these enzymes, is fairly low, especially compared to a kinase. You need to try a lot of different methods to identify your initial lead matter. Then you need a robust screening cascade to rule out all of the nonspecific molecules that you identify in the screening space. Doing that efficiently and not wasting a lot of chemistry effort on poor molecules is pretty critical. Richard T. Cummings: A couple of you have already touched on the importance of setting assays up with the appropriate substrates and/or context. In line with that, do you think that greater, or perhaps different, value is likely to be obtained if you do phenotypic screens versus more traditional, single-agent, biochemicalbased screening? Adam, please kick us off. W. Adam Hill: With respect to the last point, as well, we focus very much on the target itself. The other point I want to raise is around the toxicity and safety side of things. Obviously, if this is looking at epigenetic targets and moving them down into drug space, you have the efficacy side, but you also have the safety side. That is something we also need to come to grips with as we look at these molecules. Regarding which is likely to offer more value, again, it very much depends on the strengths and weaknesses of the library and how you go about your screening campaigns. We have a strong emphasis here
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on biophysics, so being able to do fragment-based screening and optimize through that pathway is certainly one approach. We also have some very good high-content imaging solutions to look more at the phenotypic side of things. Trying to put assays together and get strong correlations between the readouts has not always been as easy as we hoped. Having a strong flowchart to be able to progress the chemical matter is an important thing. Again, trying to get that relevance down toward the clinic is really going to be the ultimate test of how well your assays are working and being responsive to the chemical matter. So we try both. We look to see which progresses best, and then use that to leverage putting more compounds into the pipeline. Richard T. Cummings: Karen, your thoughts? Karen Maegley: I have had both good and bad experiences with phenotypic screening. Outside of the epigenetic space, I worked in antivirals for a while. We had some antiviral phenotypic screens that were very successful. In that case, the biology was much simpler. More recently, in oncology, looking at nonepigenetic signaling pathways and trying to do phenotypic screens, in my experience it becomes very, very difficult to take that hit and figure out the mechanism of how it is working to generate whatever phenotype it is that you are following. I am not a big fan of phenotypic screens. Epigenetic space brings added complexity. We spend a lot of time focusing on changes to histone modification, but a lot of these enzymes will methylate, demethylate, acetylate, or deacetylate nonhistone targets. A lot of that is emerging and not well understood. My view is that the biology might make phenotypic screening in this space even more complicated than usual. We are fairly focused on target-based approaches at this time. Richard T. Cummings: Rob, what is your perspective? Robert Sims: At Constellation, we have focused on traditional targetbased screening as a starting principle. Even from that perspective, as discussed previously, it can be challenging trying to align and correlate the different types of assays, whether biochemical or in cells. Like most screening, your success will probably hinge on the quality of your assay. In the case of antivirals, if you have a nice cell system, then that may actually provide something that is useful. It may be more challenging if it is something crude like viability. Having said all of that, we have used a hybrid approach as well, where we make small-molecule inhibitors against different types of chromatin regulators. Then we use those as tools to validate or invalidate the targets in different types of phenotypic assays. That has allowed us to quickly identify biology that we would not have otherwise. But as a starting principle, I do think that phenotypic screening, even looking at methyl or acetyl changes, will be challenging. It is more straightforward when you take a traditional target and isolate it biochemically. Richard T. Cummings: Karen, I think you were the first to touch on this, so I will ask you to address this next question first. Are there
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roles for privileged compound collections in fragment libraries for identifying optimal starting points? Are they likely to be of more value, or is there more value to be gained from larger, unbiased screening collections? What has been the experience for you and your team? Karen Maegley: I am a big fan of fragment screening, particularly for targets where finding inhibitors can be challenging, because it gives you a less-biased approach. But at least for the targets we have been working on, you have to throw everything at it. Within big pharma, the libraries do not really contain a large number of compounds that are going to be inhibitors of epigenetic-type targets, for whatever reason. We do not cover a lot of chemical space in our libraries, even though we have millions of compounds in our library. But if you take a fragment approach, or if you have a specialized library that has been rationally designed, those are two other ways, in addition to this larger unbiased screening, that are really valuable. We have to do all of these things. I really like fragment screening as a general indicator of the druggability of a particular target. The number of fragment hits you get is directly correlated to how druggable your target is. So it can give you a really quick answer on whether it is going to be a challenge. Then, perhaps that is part of your decision on whether you want to progress that target forward. It has also been said, by Adam, that you need strong biophysics if you are going to do these types of approaches, because it is critical that you have direct binding of the compound to the target. It is essential to understand the details of that binding and the location of the binding site, particularly with fragment-based screening. You must have some sort of structural biology platform in place. As long as you have those things, fragment screening is a no-brainer. Richard T. Cummings: Your thoughts, Adam? W. Adam Hill: I keep harkening back to the kinase example, which has been preceding this target class for a while. The point there is that, over time, you build up your own privileged collection of kinase inhibitors as you go off to more and more kinase targets. I think the same will be true in the epigenetic space—as you go off to more and more targets, you will build up your own privileged collection and look for opportunities to jump from one target to the next. That will jump-start things. We applied fragment libraries to some of these targets, and with the strong biophysics that really helps. The caveat is that we tend to make some sort of compromise in the protein or the target we are using. By truncating the protein, you are leaving out domains. You are leaving out other potential sites that you could go after. If you are going for a very traditional, catalytic domain-type inhibitor, I think a fragment-based approach is usually very appropriate. When going after other domains, you must be very careful as to how you construct your screens. You need to make sure that you are finding the relevant chemical matter, which will then translate well into further assays as you continue the medicinal chemistry optimization. Much of this has to be done in close collaboration with medicinal chemistry to work out some of the wrinkles.
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We have seen many occasions where the activity of the compounds has been very nonspecific. This illustrates the importance of making sure that your library collection is clean, that you are transition metal-free, and that you are really following the activity of the molecules as they are identified in the library, as opposed to anything else that may be coming along with them. Richard T. Cummings: Rob, your perspective? Robert Sims: Karen and Adam highlighted the key points. At Constellation, we have had success with privileged collections, fragment screening, as well as screening large collections in an unbiased way and finding chemical matter that may have been really challenging to find if we were more focused. So, thinking about the target is important. Certainly, for a class like the bromodomains, which are beautiful drug discovery targets from a protein and a biochemistry/ biophysics perspective, crystallography is fantastic. And so, with bromodomains, fragment screening has also been very fruitful. Again, going back to an earlier point, you can tell who has had experience in this space, harping on good validation biophysical tools. But many of these targets function in large, multiprotein complexes. Certainly, in the context of readers, sometimes these complexes have multiple functions—catalytic activity, hydrolyzed ATP, and demobilized nucleosomes—and it creates challenges when trying to understand how blocking or perturbing one protein–protein interaction may impact the enzymology of a variety of different readouts within that complex. To reconstitute that and understand it in vitro is challenging. It puts constraints on what you can do biochemically and biophysically. But it is highly relevant in understanding how that pharmacologic inhibition is having an impact on not just the one protein or domain, but the entire complex that you are actually inhibiting in the cell. Richard T. Cummings: Rob, you were the first to touch on this, so I will ask you to comment again right away. One thing that has emerged in this field is that these epigenetic enzymes often have comparatively low catalytic activity and correspondingly slow changes in the turnover of the epigenetic marks, in the context of both cells and organisms. Does this create additional challenges, and what sort of things need to be considered in this context? Robert Sims: It creates all kinds of challenges, such as trying to faithfully recapitulate that biochemical activity in a tube and to faithfully capture that in your screen. Karen spoke earlier about proper reconstitution of chromatin templates. Is it recombinant, or do we use native chromatin? Is it mononucleosomes? Do you want to add magnesium to compact or not compact? You see altering activity when you vary the flavor of any of those parameters. You add an activating peptide for PRC2, and you see a stimulatory activity. All of those components add to the complexity. Making sure that you can faithfully recapitulate that activity in your screen will always be a question with these enzymes. Once we have been lucky enough to have chemical matter and cell-based activity, and after having seen that turnover manifest
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slowly over time, we have used a lot of histone mass spectrometry to understand the dynamics of methylation turnover through inhibiting a number of different pathways. The complexity is humbling. It is important to understand that, especially as it relates to a phenotype that may or may not occur. It is just something, especially with methyltransferases, that you need to pay attention to. On the other end of the spectrum are targets like bromodomains, where literally within 10 minutes you can have complete eviction of your target from chromatin. Understanding the relationship between those two bookends and how it relates to your program is really important. Richard T. Cummings: Karen? Karen Maegley: A couple of things. First, from the biochemistry side, it does put a lot of pressure on the assay. One thing we have done is focus on assay formats that directly monitor the product, in as labelfree an environment as possible. For example, we use a lot of mass spectrometry just as a plate reader for biochemistry assays, not to do any of the fancy stuff like monitoring different combinatorial changes, which is also very useful, but from a biochemical assay perspective, using the change in mass as a way to detect your product directly and not using a bunch of coupling enzymes or colorimetric readouts where you run into a lot of background activities and issues with assay formats. We have some very specific assays with very low backgrounds that allow us to detect very, very few rounds of turnover above backgrounds highly quantitatively, and very reproducibly. That is a big thing. The other thing is more of a mentality. For example, with methyltransferases, it takes several days for the methyl mark to change in cells, and then 7–14 days to see the phenotype in a cell-based assay. There are many with kinase backgrounds who are expecting things to happen a lot faster, and so there is a lot of concern about whether or not the pharmacology that you are observing is directly due to inhibition of the target that you are going after. There is a lot of extra work required to prove that the compound is working on the target, and that the target is causing the change that you are observing. As we get more experience working with these inhibitors, that will become easier. Everywhere down the line, it adds a challenge. You have to do in vivo experiments for longer periods of time, which creates its own problems with setting up your models. It requires synthesis of a larger quantity of compound. Hence, there are challenges at every step along the path. Richard T. Cummings: Adam, your perspective? W. Adam Hill: Rob and Karen have raised some very good points. We too use a lot of mass spectrometry early in primary screening to look for activity. Again, trying to deconvolute how particular molecules can interrupt different parts of, say, the methyltransferases—whether it stops at a mono-, di-, or tri-methyl—is a challenge. The other point is that many of these enzymes have a number of different functions. It is important to not necessarily just follow
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activity, but also have ways to robustly measure things like protein– protein interactions. Another point here about the delayed growth in cell-based assays is that it is certainly a challenge that we had to get our heads around in the screening group, to run assays beyond, say, 5 days. Being able to run a reasonable number of compounds when doing a 10-day assay, keeping the sterility, making sure that everyone is fed and watered appropriately, and so on, are some challenges to putting those assays together. Again, with any new target class, you have to invent some new toys and new techniques to be able to get the job done. In the case of epigenetics, there is no difference. Richard T. Cummings: We have one question left. Stepping back up to a bit of a higher level as we bring this to a conclusion, I will ask all of the participants for their longer-range view of the field of epigenetic drug discovery, specifically looking out 5–10 years. Where do you see epigenetic drug discovery and its downstream clinical applications? Will they be growing? Are there additional epigenetic classes likely to be exploited beyond the current ones? What else should be in place to facilitate this? Karen, I will ask for your perspective first. Karen Maegley: Hopefully, in 5–10 years, we are where kinases are now. I hope that we have built up privileged libraries and can more readily find chemical matter, and I hope that we understand the biology that much better. What I would like to see happen in the next 5–10 years is a much better understanding of how to go after more of the solid tumors. There has been a fair amount of success recently with hematologic malignancies. We will need to expand outside of that if we are going to continue to be successful and continue to bring forward new epigenetic therapies. As far as moving beyond bromodomains and methyltransferase inhibitors, I am confident we will. I believe we will find a way to bring forward the demethylases and others, perhaps even acetyltransferases, as well. This is a very new, young, emerging field. There are a lot of really smart people working in this space and a lot of really cool science being done. I feel that it is going to become something very powerful, and have impact beyond oncology as well. I am excited for the future. Richard T. Cummings: Adam, any additional comments? W. Adam Hill: I will begin with my doom and gloom, and hopefully end on a joyful note. With the targets that have come through to date, there is good genetic validation. I think the low-hanging fruit has been picked over fairly well. Going forward, the question is, are there more genetically validated targets that we can go after? With a lot of the fundamental
research going on, what I am hoping is that we will understand much more about how these epigenetic mechanisms can be modulated, which will in turn provide the next round of valid targets for us to go after. At the moment, most people are focused on oncology. But I see an epigenetic role in many other disease areas too. By combining an understanding of the safety profiles that are needed, as well as an understanding of the mechanisms by which particular targets are modulated, I can envision that in 10 years this will be as big as—if not bigger than—the kinases have been up to now. Richard T. Cummings: Rob, what are your final thoughts? Robert Sims: From the clinical viewpoint, we will see how it plays out from a safety perspective. I think if there are signs of single-agent activity—I will pick on BET and EZH2, as those two are out in front— you will very likely see the application grow, because it is such a different modality. The biology that is being touched by those two different targets is relatively unique to what is currently being used therapeutically. I think you will see a lot of activity in combination, and that is both in hematologic malignancies as well as in epithelial-derived cancers. Hopefully, that clinical application will grow, again, depending on the safety and tolerability of this first wave of compounds. Some of that clinical data will be the rheostat of excitement for the next wave, because I agree with Adam that it will be more challenging. These first targets are complex enough. Then you start trying to exploit the many different types of epigenetic mutations that are found, the preponderance of which are inactivating mutations. The next wave of targets will be more challenging, but it may end up being just as rewarding. But it will take a commitment to the space. It will mean embracing the complexity of the biochemistry and the biology, and working with the academic community to understand how these targets function and are mechanistically yielding the phenotypes that we think are therapeutically relevant. Despite a lot of challenges, it is a very exciting space to be in. It takes some bravery to keep pushing forward. Richard T. Cummings: With that, I will bring this roundtable to a close. I have enjoyed everyone’s perspective. There are general themes we have heard across the table, that there are both significant opportunities and also very significant challenges. Over the next decade or so, the field of epigenetic drug discovery promises to be very exciting.
DISCLOSURE STATEMENT None of the participants of this roundtable have any financial conflicts to disclose.
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Challenges and Opportunities in Epigenetic Drug Discovery Moderator: Richard T. Cummings1 Participants: Karen Maegley,2 W. Adam Hill,3 Robert Sims,4 and Sophie Mohin5 1
Senior Director, Lead Discovery, and 4Senior Director of Biology, Constellation Pharmaceuticals, Cambridge, Massachusetts. 2 Associate Research Fellow, Oncology Research Unit, Pfizer, San Diego, California. 3 Director, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts. 5 Assistant Managing Editor, Mary Ann Liebert, Inc., New Rochelle, New York.
Richard T. Cummings: Welcome to this roundtable discussion on epigenetic drug discovery. We have three participants: Karen Maegley from Pfizer in San Diego; Adam Hill from Novartis in Cambridge, Massachusetts; and Rob Sims from Constellation Pharmaceuticals in Cambridge, Massachusetts. Let us start with a general question, and then drill down a bit. We will begin with the question: What is your view on the current state of epigenetic drug discovery? Rob, please kick it off. Robert Sims: I will take a historical look back, at least from our perspective at Constellation Pharmaceuticals. We have been around for a little more than six years. When we were discussing the outlook for epigenetic drug discovery with our venture-backed support, several people questioned whether it was the right time or if it was too early. There were a number of companies that had already invested in epigenetics, for example, Novartis, because of the potential application in a variety of therapeutic areas. If you fast-forward six years later, I think the community as a whole, both in industry and in academia, has made tremendous progress. Perhaps the most important recent progress has been the observation of multiple epigenetic modalities that are beginning to show hints of clinical activity. From that perspective alone, there has probably never been a more exciting time to be in the epigenetic space. That is interesting from a number of different paradigms: writers, readers, as well as targets like IDH1 and IDH2, which very clearly impact the epigenetic state of cancers that have mutations in those targets and very robustly affect the methylation state of cells. Again, this reveals very interesting early signs of clinical activity. It is very exciting, and I think the next question at a high level is: Will this be sustainable over the next 5–10 years?
DOI: 10.1089/adt.2014.1502
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Richard T. Cummings: Karen? Karen Maegley: I am relatively new to this space compared to my colleagues at Constellation. I have been working in the epigenetic space for only a couple of years. But I completely agree with what was just said. In the early days (even from my perspective, which was late days from the Constellation perspective), there were many questions about whether we were getting in too early and whether we knew enough about the biology to really know which targets to choose. The industry as a whole (Epizyme, Constellation, GlaxoSmithKline, and others) has helped us in terms of the general acceptance of epigenetics as a therapy—for us, specifically in oncology, which is where I work. It is a really exciting time because it is still very new, and we do not know a lot about how to actually use these molecules. But we have definitely come a long way. Richard T. Cummings: And lastly, Adam? W. Adam Hill: Epigenetics is an emerging field. If you thought about regulating proteins and their activity 20-plus years ago, you would have been talking about kinases. There were some good breakthroughs in that area, which led to a large number of companies focusing on kinases as targets. I see epigenetic targets as one of the next generation of target classes, to be able to regulate proteins within the cell. From that perspective, I think there are some proven examples. There is a lot of chemical matter going through the clinic. Behind that, there is a lot of fundamental research going on. I see epigenetics as a burgeoning target class, which over the next few years will hopefully lead to drugs that make a real impact in patients’ lives. Richard T. Cummings: Following up on Adam’s comment about this being an emerging class, are there challenges associated with epigenetic targets that are unique to them relative to other, more traditional classes, such as G-protein coupled receptors (GPCRs) or kinases? If there are challenges, how have your teams addressed them? Rob, please start us off on this. Robert Sims: There are many challenges associated with the space, although, in general, most areas have challenges that are unique to them. You are planning to touch on this a little bit later, so I will skip the component about enzyme kinetics and the delayed response in cells. That is highly relevant to the epigenetic space when trying to monitor target engagement as well as phenotypic responses. Understanding how those molecular changes are manifested over time has been challenging, and it is something that the field has made progress on. I will use that as one example of the challenges and complexity of the epigenetic space.
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My scientific background has been rooted in chromatin and transcription. As we entered this particular field from a drug discovery perspective, like most things, we underestimated the complexity that would be visualized once we began to selectively and potently inhibit some of these chromatin coregulators. Epigenetic targets are not like kinases, for example, where you have an inhibitor and it inhibits catalytic activity (e.g., phosphorylation) of a particular kinase target. That is something you can rely on, although obviously there are complexities around that. What we found with bromodomain and extra terminal (BET) is that if you look in different cell contexts, the localization or the pattern of BET binding throughout the genome is quite different from cell to cell. Moreover, when you change or stimulate that cell, even in a one-cell context, the localization and distribution of that protein changes, and it can change over time. We have done experiments looking at very early transcriptional changes, minutes after treatment with BET inhibitors. That response is very dynamic over time, and it redistributes over time. In a way, it is a moving target. Trying to understand how inhibition of chromatin binding impacts a transcriptional program, even in one context over many dimensions, is complicated. But then trying to translate that into something practical, like how do I define predictive biomarkers of response in a patient population, just underscores the complexity and the challenge of dealing with these types of targets in this particular field. We and others in the field have made progress in understanding that, but it is absolutely a challenge when thinking about modulators of oncogenes as opposed to targeting oncogenes themselves. It is great that we know about the activating mutations of EZH2, but it has activity outside of that. Understanding that, how do you identify the populations that will best benefit from that mechanism? That is a challenge that obviously will have to be addressed over the next few years in trying to make these particular therapies useful to patients. Richard T. Cummings: Adam? W. Adam Hill: Rob touched on a number of good points. One difficulty we found when we first entered this field was working out exactly what a relevant assay was. You could go for a very simplistic form, for example, just looking at a catalytic activity. But when trying to put that into context and understand the different modes by which you could actually interrupt the activity, the numbers started to get much larger, and the ability to prove which assay was giving you the relevant information was quite tricky. So challenges include picking the target and working out how to make a biochemical assay for it, or looking at a cell-based assay and determining what an appropriate readout for that would be. Even if you are going into a phenotype, it is a challenge trying to define what that phenotype should be, thinking about three-dimensional cell culture versus two-dimensional, things like that. I think they all play into getting some more fundamental understanding of the biology around these targets, which allows you to put the appropriate assays together. With GPCRs or kinases, we have been in the field long enough to have a good handle on some of the quirks and nuances around those
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target families. Going into a new space like epigenetics, we have to come up, again, with a new toolkit, a new set of ways to go after these targets. Richard T. Cummings: Karen, have you found your experience in line with the other participants’? Karen Maegley: Absolutely. To expand on a couple of those things, with respect to a pharmacodynamic biomarker, for example, in a kinase, it is pretty straightforward to set up a cell-based assay to look at something like autophosphorylation or phosphorylation of the next kinase down the signaling pathway. But for some of these epigenetic targets, for example, a methyltransferase, monitoring the changes in the methyl mark at a global level will not always give you the information you need for your specific target. Sometimes it does and sometimes it doesn’t. Just figuring out what your cell-based readout is going to be can be quite challenging, depending on the target. Going back to the comment about a biologically relevant assay, if you want to use a nucleosome as a substrate, for example, it can be difficult to prepare with the quality that is necessary to do good biochemistry. If you are not using recombinant mononucleosomes, the oligonucleosome structure is very dynamic and very changeable, depending on what additives are in your assay buffer. Those are things that you really need to pay attention to. Using a peptide substrate is frequently not a very good surrogate, in my opinion, for a methyltransferase substrate. But from the biochemistry side of things, druggability seems to be a huge problem for us. With the BET inhibitors, that has not been an issue, but it seems to me, particularly in the methyltransferase space, that the number of solutions, the number of molecules that we have in our various collections that will be real inhibitors of these enzymes, is fairly low, especially compared to a kinase. You need to try a lot of different methods to identify your initial lead matter. Then you need a robust screening cascade to rule out all of the nonspecific molecules that you identify in the screening space. Doing that efficiently and not wasting a lot of chemistry effort on poor molecules is pretty critical. Richard T. Cummings: A couple of you have already touched on the importance of setting assays up with the appropriate substrates and/or context. In line with that, do you think that greater, or perhaps different, value is likely to be obtained if you do phenotypic screens versus more traditional, single-agent, biochemicalbased screening? Adam, please kick us off. W. Adam Hill: With respect to the last point, as well, we focus very much on the target itself. The other point I want to raise is around the toxicity and safety side of things. Obviously, if this is looking at epigenetic targets and moving them down into drug space, you have the efficacy side, but you also have the safety side. That is something we also need to come to grips with as we look at these molecules. Regarding which is likely to offer more value, again, it very much depends on the strengths and weaknesses of the library and how you go about your screening campaigns. We have a strong emphasis here
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on biophysics, so being able to do fragment-based screening and optimize through that pathway is certainly one approach. We also have some very good high-content imaging solutions to look more at the phenotypic side of things. Trying to put assays together and get strong correlations between the readouts has not always been as easy as we hoped. Having a strong flowchart to be able to progress the chemical matter is an important thing. Again, trying to get that relevance down toward the clinic is really going to be the ultimate test of how well your assays are working and being responsive to the chemical matter. So we try both. We look to see which progresses best, and then use that to leverage putting more compounds into the pipeline. Richard T. Cummings: Karen, your thoughts? Karen Maegley: I have had both good and bad experiences with phenotypic screening. Outside of the epigenetic space, I worked in antivirals for a while. We had some antiviral phenotypic screens that were very successful. In that case, the biology was much simpler. More recently, in oncology, looking at nonepigenetic signaling pathways and trying to do phenotypic screens, in my experience it becomes very, very difficult to take that hit and figure out the mechanism of how it is working to generate whatever phenotype it is that you are following. I am not a big fan of phenotypic screens. Epigenetic space brings added complexity. We spend a lot of time focusing on changes to histone modification, but a lot of these enzymes will methylate, demethylate, acetylate, or deacetylate nonhistone targets. A lot of that is emerging and not well understood. My view is that the biology might make phenotypic screening in this space even more complicated than usual. We are fairly focused on target-based approaches at this time. Richard T. Cummings: Rob, what is your perspective? Robert Sims: At Constellation, we have focused on traditional targetbased screening as a starting principle. Even from that perspective, as discussed previously, it can be challenging trying to align and correlate the different types of assays, whether biochemical or in cells. Like most screening, your success will probably hinge on the quality of your assay. In the case of antivirals, if you have a nice cell system, then that may actually provide something that is useful. It may be more challenging if it is something crude like viability. Having said all of that, we have used a hybrid approach as well, where we make small-molecule inhibitors against different types of chromatin regulators. Then we use those as tools to validate or invalidate the targets in different types of phenotypic assays. That has allowed us to quickly identify biology that we would not have otherwise. But as a starting principle, I do think that phenotypic screening, even looking at methyl or acetyl changes, will be challenging. It is more straightforward when you take a traditional target and isolate it biochemically. Richard T. Cummings: Karen, I think you were the first to touch on this, so I will ask you to address this next question first. Are there
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roles for privileged compound collections in fragment libraries for identifying optimal starting points? Are they likely to be of more value, or is there more value to be gained from larger, unbiased screening collections? What has been the experience for you and your team? Karen Maegley: I am a big fan of fragment screening, particularly for targets where finding inhibitors can be challenging, because it gives you a less-biased approach. But at least for the targets we have been working on, you have to throw everything at it. Within big pharma, the libraries do not really contain a large number of compounds that are going to be inhibitors of epigenetic-type targets, for whatever reason. We do not cover a lot of chemical space in our libraries, even though we have millions of compounds in our library. But if you take a fragment approach, or if you have a specialized library that has been rationally designed, those are two other ways, in addition to this larger unbiased screening, that are really valuable. We have to do all of these things. I really like fragment screening as a general indicator of the druggability of a particular target. The number of fragment hits you get is directly correlated to how druggable your target is. So it can give you a really quick answer on whether it is going to be a challenge. Then, perhaps that is part of your decision on whether you want to progress that target forward. It has also been said, by Adam, that you need strong biophysics if you are going to do these types of approaches, because it is critical that you have direct binding of the compound to the target. It is essential to understand the details of that binding and the location of the binding site, particularly with fragment-based screening. You must have some sort of structural biology platform in place. As long as you have those things, fragment screening is a no-brainer. Richard T. Cummings: Your thoughts, Adam? W. Adam Hill: I keep harkening back to the kinase example, which has been preceding this target class for a while. The point there is that, over time, you build up your own privileged collection of kinase inhibitors as you go off to more and more kinase targets. I think the same will be true in the epigenetic space—as you go off to more and more targets, you will build up your own privileged collection and look for opportunities to jump from one target to the next. That will jump-start things. We applied fragment libraries to some of these targets, and with the strong biophysics that really helps. The caveat is that we tend to make some sort of compromise in the protein or the target we are using. By truncating the protein, you are leaving out domains. You are leaving out other potential sites that you could go after. If you are going for a very traditional, catalytic domain-type inhibitor, I think a fragment-based approach is usually very appropriate. When going after other domains, you must be very careful as to how you construct your screens. You need to make sure that you are finding the relevant chemical matter, which will then translate well into further assays as you continue the medicinal chemistry optimization. Much of this has to be done in close collaboration with medicinal chemistry to work out some of the wrinkles.
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We have seen many occasions where the activity of the compounds has been very nonspecific. This illustrates the importance of making sure that your library collection is clean, that you are transition metal-free, and that you are really following the activity of the molecules as they are identified in the library, as opposed to anything else that may be coming along with them. Richard T. Cummings: Rob, your perspective? Robert Sims: Karen and Adam highlighted the key points. At Constellation, we have had success with privileged collections, fragment screening, as well as screening large collections in an unbiased way and finding chemical matter that may have been really challenging to find if we were more focused. So, thinking about the target is important. Certainly, for a class like the bromodomains, which are beautiful drug discovery targets from a protein and a biochemistry/ biophysics perspective, crystallography is fantastic. And so, with bromodomains, fragment screening has also been very fruitful. Again, going back to an earlier point, you can tell who has had experience in this space, harping on good validation biophysical tools. But many of these targets function in large, multiprotein complexes. Certainly, in the context of readers, sometimes these complexes have multiple functions—catalytic activity, hydrolyzed ATP, and demobilized nucleosomes—and it creates challenges when trying to understand how blocking or perturbing one protein–protein interaction may impact the enzymology of a variety of different readouts within that complex. To reconstitute that and understand it in vitro is challenging. It puts constraints on what you can do biochemically and biophysically. But it is highly relevant in understanding how that pharmacologic inhibition is having an impact on not just the one protein or domain, but the entire complex that you are actually inhibiting in the cell. Richard T. Cummings: Rob, you were the first to touch on this, so I will ask you to comment again right away. One thing that has emerged in this field is that these epigenetic enzymes often have comparatively low catalytic activity and correspondingly slow changes in the turnover of the epigenetic marks, in the context of both cells and organisms. Does this create additional challenges, and what sort of things need to be considered in this context? Robert Sims: It creates all kinds of challenges, such as trying to faithfully recapitulate that biochemical activity in a tube and to faithfully capture that in your screen. Karen spoke earlier about proper reconstitution of chromatin templates. Is it recombinant, or do we use native chromatin? Is it mononucleosomes? Do you want to add magnesium to compact or not compact? You see altering activity when you vary the flavor of any of those parameters. You add an activating peptide for PRC2, and you see a stimulatory activity. All of those components add to the complexity. Making sure that you can faithfully recapitulate that activity in your screen will always be a question with these enzymes. Once we have been lucky enough to have chemical matter and cell-based activity, and after having seen that turnover manifest
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slowly over time, we have used a lot of histone mass spectrometry to understand the dynamics of methylation turnover through inhibiting a number of different pathways. The complexity is humbling. It is important to understand that, especially as it relates to a phenotype that may or may not occur. It is just something, especially with methyltransferases, that you need to pay attention to. On the other end of the spectrum are targets like bromodomains, where literally within 10 minutes you can have complete eviction of your target from chromatin. Understanding the relationship between those two bookends and how it relates to your program is really important. Richard T. Cummings: Karen? Karen Maegley: A couple of things. First, from the biochemistry side, it does put a lot of pressure on the assay. One thing we have done is focus on assay formats that directly monitor the product, in as labelfree an environment as possible. For example, we use a lot of mass spectrometry just as a plate reader for biochemistry assays, not to do any of the fancy stuff like monitoring different combinatorial changes, which is also very useful, but from a biochemical assay perspective, using the change in mass as a way to detect your product directly and not using a bunch of coupling enzymes or colorimetric readouts where you run into a lot of background activities and issues with assay formats. We have some very specific assays with very low backgrounds that allow us to detect very, very few rounds of turnover above backgrounds highly quantitatively, and very reproducibly. That is a big thing. The other thing is more of a mentality. For example, with methyltransferases, it takes several days for the methyl mark to change in cells, and then 7–14 days to see the phenotype in a cell-based assay. There are many with kinase backgrounds who are expecting things to happen a lot faster, and so there is a lot of concern about whether or not the pharmacology that you are observing is directly due to inhibition of the target that you are going after. There is a lot of extra work required to prove that the compound is working on the target, and that the target is causing the change that you are observing. As we get more experience working with these inhibitors, that will become easier. Everywhere down the line, it adds a challenge. You have to do in vivo experiments for longer periods of time, which creates its own problems with setting up your models. It requires synthesis of a larger quantity of compound. Hence, there are challenges at every step along the path. Richard T. Cummings: Adam, your perspective? W. Adam Hill: Rob and Karen have raised some very good points. We too use a lot of mass spectrometry early in primary screening to look for activity. Again, trying to deconvolute how particular molecules can interrupt different parts of, say, the methyltransferases—whether it stops at a mono-, di-, or tri-methyl—is a challenge. The other point is that many of these enzymes have a number of different functions. It is important to not necessarily just follow
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activity, but also have ways to robustly measure things like protein– protein interactions. Another point here about the delayed growth in cell-based assays is that it is certainly a challenge that we had to get our heads around in the screening group, to run assays beyond, say, 5 days. Being able to run a reasonable number of compounds when doing a 10-day assay, keeping the sterility, making sure that everyone is fed and watered appropriately, and so on, are some challenges to putting those assays together. Again, with any new target class, you have to invent some new toys and new techniques to be able to get the job done. In the case of epigenetics, there is no difference. Richard T. Cummings: We have one question left. Stepping back up to a bit of a higher level as we bring this to a conclusion, I will ask all of the participants for their longer-range view of the field of epigenetic drug discovery, specifically looking out 5–10 years. Where do you see epigenetic drug discovery and its downstream clinical applications? Will they be growing? Are there additional epigenetic classes likely to be exploited beyond the current ones? What else should be in place to facilitate this? Karen, I will ask for your perspective first. Karen Maegley: Hopefully, in 5–10 years, we are where kinases are now. I hope that we have built up privileged libraries and can more readily find chemical matter, and I hope that we understand the biology that much better. What I would like to see happen in the next 5–10 years is a much better understanding of how to go after more of the solid tumors. There has been a fair amount of success recently with hematologic malignancies. We will need to expand outside of that if we are going to continue to be successful and continue to bring forward new epigenetic therapies. As far as moving beyond bromodomains and methyltransferase inhibitors, I am confident we will. I believe we will find a way to bring forward the demethylases and others, perhaps even acetyltransferases, as well. This is a very new, young, emerging field. There are a lot of really smart people working in this space and a lot of really cool science being done. I feel that it is going to become something very powerful, and have impact beyond oncology as well. I am excited for the future. Richard T. Cummings: Adam, any additional comments? W. Adam Hill: I will begin with my doom and gloom, and hopefully end on a joyful note. With the targets that have come through to date, there is good genetic validation. I think the low-hanging fruit has been picked over fairly well. Going forward, the question is, are there more genetically validated targets that we can go after? With a lot of the fundamental
research going on, what I am hoping is that we will understand much more about how these epigenetic mechanisms can be modulated, which will in turn provide the next round of valid targets for us to go after. At the moment, most people are focused on oncology. But I see an epigenetic role in many other disease areas too. By combining an understanding of the safety profiles that are needed, as well as an understanding of the mechanisms by which particular targets are modulated, I can envision that in 10 years this will be as big as—if not bigger than—the kinases have been up to now. Richard T. Cummings: Rob, what are your final thoughts? Robert Sims: From the clinical viewpoint, we will see how it plays out from a safety perspective. I think if there are signs of single-agent activity—I will pick on BET and EZH2, as those two are out in front— you will very likely see the application grow, because it is such a different modality. The biology that is being touched by those two different targets is relatively unique to what is currently being used therapeutically. I think you will see a lot of activity in combination, and that is both in hematologic malignancies as well as in epithelial-derived cancers. Hopefully, that clinical application will grow, again, depending on the safety and tolerability of this first wave of compounds. Some of that clinical data will be the rheostat of excitement for the next wave, because I agree with Adam that it will be more challenging. These first targets are complex enough. Then you start trying to exploit the many different types of epigenetic mutations that are found, the preponderance of which are inactivating mutations. The next wave of targets will be more challenging, but it may end up being just as rewarding. But it will take a commitment to the space. It will mean embracing the complexity of the biochemistry and the biology, and working with the academic community to understand how these targets function and are mechanistically yielding the phenotypes that we think are therapeutically relevant. Despite a lot of challenges, it is a very exciting space to be in. It takes some bravery to keep pushing forward. Richard T. Cummings: With that, I will bring this roundtable to a close. I have enjoyed everyone’s perspective. There are general themes we have heard across the table, that there are both significant opportunities and also very significant challenges. Over the next decade or so, the field of epigenetic drug discovery promises to be very exciting.
DISCLOSURE STATEMENT None of the participants of this roundtable have any financial conflicts to disclose.
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